The objective of this project is to develop robust assays that will allow real-time monitoring and quantification of specific alternative splicing events in living cells, and to adapt them for high throughput screening (HTS) against libraries of small compounds. Deregulation of alternative splicing patterns is frequently associated with several neurodegenerative and other genetic diseases and is a common occurrence in most cancers. The ability to re-direct splicing events to correct such splicing defects or to force the expression of alternative splicing isoform with potential beneficial effects would provide a valuable tool to investigate the roles and functions of specific alternative splicing isoforms, and it will open wide possibilities for novel therapeutic approaches. We plan to develop a set of minigene constructs in which the expression of a transiently transfected fluorescent reporter protein will quantitatively depend on a specific splicing event. The initial testing will investigate alternative splicing events in STAT3 and BRCA1, but the setup will be readily transferable to different systems. The assay will be initially performed in 96 well plates and used in low-to-medium throughput experiments to identify and characterize cis-elements and trans acting factors that regulate these splicing events, by means of systematic over-expression and/or knockdown of splicing factors combined with substrate mutagenesis. Multiple fluorescent variants will be tested in various combinations using the Amersham INCell analyzer, to eventually develop a multicolor protocol to simultaneously assess different splicing events in individual cells, along with appropriate counter screenings and validation procedures. The protocol will be optimized to 384 well microtiter plates and will be suitable to HTS of large small molecule libraries.